The long term objective of this project is to develop technology to provide low-cost (<$1/bp), rapid synthesis of complete genes (500 bp to 5 kb). Rapid, accurate and affordable gene synthesis will provide a direct link between genomic information and hypothesis-driven experimental science, improving our ability to understand disease processes and design therapeutic agents. Examples of the ways in which gene synthesis can impact health-related projects include codon optimization, splice variant construction and protein engineering for expression of human and pathogen proteins in heterologous hosts for structural genomics, antigen production and use as targets / reporters for drug discovery efforts. Gene synthesis is currently too slow and expensive to meet the needs of the genomic and health sciences community. The lowest prices are around $4/bp with turnaround times of 4-8 weeks for a gene of 1 kb. The major factors governing price are the costs of building block synthesis and sequence confirmation. The specific aim of this project is to develop methods and machines to synthesize 1-2 pmol of oligonucleotides with isolated purity > 90% and to reliably assemble these into genes. We aim to lower the cost of building block synthesis 3- to 10-fold by combining advanced chemistry and alternative engineering to reduce reaction volumes and reagent concentrations. We will also optimize synthesis on a 2-dimensional support to improve the stepwise coupling efficiency to >99.5%. Small scale, low cost, high coupling efficiency oligonucleotide synthesis will allow greater flexibility in oligonucleotide gene building block design, remove the need for oligonucleotide purification prior to assembly into complete genes, reduce sequence confirmation costs and shorten order to delivery times. To achieve this, in Phase 1 we will develop analytical methods to measure oligonucleotide synthesis quality, construct a manual device for synthesis of oligonucleotides on a non-porous glass support, determine efficiency-limiting steps and demonstrate ability to produce oligonucleotides with >90% purity and show that they can be used directly for high fidelity gene synthesis. A low cost high fidelity oligonucleotide manufacturing process may have additional applications in related areas including single nucleotide polymorphism detection, expression array manufacture and chip-based sequencing.